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Journal of Lipid Research, Vol. 43, 2196-2204, December 2002
Copyright © 2002 by Lipid Research, Inc.

Methods

Novel d--tocopherol derivative as a prodrug for d--tocopherol and a two-step prodrug for S--CEHC

Jiro Takata^1,*, Ryoji Hidaka^*, Akihiko Yamasaki^*, Akihiro Hattori, Takeshi Fukushima, Maiko Tanabe, Kazuhisa Matsunaga^*, Yoshiharu Karube^* and Kazuhiro Imai

^* Faculty of Pharmaceutical Sciences, Fukuoka University, Nanakuma, Johnan-ku, Fukuoka 814-0180, Japan
Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Published, JLR Papers in Press, September 16, 2002. DOI 10.1194/jlr.D200027-JLR200

¹ To whom correspondence should be addressed. e-mail: jtakata{at}fukuoka-u.ac.jp

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
d--Tocopherol (-Toc) and its major metabolite, 2, 7, 8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC), are currently receiving attention concerning their unique pharmacological activities. In order to achieve the efficient delivery of -Toc and S--CEHC in vivo, we synthesized d--tocopheryl N,N-dimethylglycinate hydrochloride (-TDMG) as a water-soluble prodrug of -Toc and a two-step prodrug of S--CEHC. -TDMG is a solid (mp 161–163°C) and is quite soluble in water over 50 mM. The hydrolysis of -TDMG was effectively catalyzed by esterases in rat and human liver microsomes. The disposition of -TDMG after iv administration in rats was compared with that of -Toc solubilized with the surfactant, polyoxyethylene hydrogenated castor oil. The plasma and liver levels of -Toc rapidly increased after the iv administration of the -TDMG. The liver availability of -Toc after the administration of -TDMG was two times higher than that of the -Toc administration. The relative systemic availability of S--CEHC after the -TDMG administration was an equivalent value (102%), and the mean residence time of S--CEHC was eight times longer than the racemic -CEHC administration.

Based on these results, -TDMG was identified as the most promising water-soluble prodrug of -Toc and the two-step prodrug of S--CEHC.

Abbreviations: AUC, area under the concentration-time curve; DBD-PZ, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole; FD-MS, field desorption mass spectrometry; HCO-60, polyoxyethylene hydrogenated castor oil; ¹H-NMR, proton nuclear magnetic resonance spectrometry; -Toc, d--tocopherol; -TDMG, d--tocopheryl N,N-dimethylglycinate hydrochloride; MRT, mean residence time; R--CEHC, 2,7,8-trimethyl-2R-(ß-carboxyethyl)-6-hydroxychroman; S--CEHC, 2,7,8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman; TFA, trifluoroacetic acid

Supplementary key words water-soluble prodrug • drug delivery • d--tocopherol • two-step prodrug

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
d--Tocopherol (2R,4'R,8'R--Tocopherol, -Toc) is one of the major forms of natural tocopherols (vitamin E) and constitutes 70–80% of the vitamin E in the diets of people in the United States (1). For many years, d--tocopherol (2R,4'R,8'R--Tocopherol, -Toc) was generally considered as the most active vitamin E and -Toc was mostly ignored because of its poor bioactivity as defined by the rat fetal resorption assay (2). However, -Toc is currently receiving attention concerning its beneficial effects. Several independent investigations have demonstrated that the plasma concentration of -Toc, not of -Toc, was correlated to the incidence of coronary heart disease (3–5). -Toc is superior to -Toc in its ability to trap reactive nitrogen species, mutagenic electrophiles generated during inflammation (6–9). Recently, it has been shown that -Toc was efficiently metabolized to 2,7,8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC, S-LLU-), and S--CEHC exhibited a natriuretic activity (10–12). In addition, it has been shown that -Toc and S--CEHC inhibited the generation of prostaglandin E₂ (PGE₂), an important mediator synthesized via the cyclooxygenase-2 (COX-2)-catalyzed oxidation of arachidonic acid during inflammation (13). Thus, -Toc and its metabolite, S--CEHC, are also expected to exhibit important pharmacological activities as a drug.

Upon considering the therapeutic formulations of -Toc, we have to overcome the unavoidable problem that -Toc is a highly viscous oil, practically insoluble in water and readily oxidized by atmospheric oxygen. These physicochemical properties of -Toc limit its therapeutic applications, making difficult an efficient administration of -Toc to patients. Besides, S--CEHC is also readily oxidizable as -Toc and the bioavailability of S--CEHC is very low due to its rapid elimination rate (12, 14). Thus, it can be expected that an effective delivery of -Toc would be a meaningful method for achieving the adequate bioavailability of S--CEHC. When administered in the form of an oil solution or some kind of oil emulsion, lipophilic compounds usually show a poor bioavailability regardless of their administration routes (e.g., parenteral, oral, or topical). In order to solubilize -Toc in water, a large amount of surfactant [e.g., polyoxyethylene hydrogenated castor oil (HCO-60)] would be needed for the solution formulation. The use of surfactants is, however, undesirable for a parenteral dosage because it generally induces toxicity such as an anaphylactoid reaction.

It is well known that the prodrug approach is a useful approach to improve the physicochemical properties of parent drugs. The phenolic functional group in -Toc is easily esterified and some of the ester derivatives are expected to provide the desired improvements in water-solubility and the stability to oxidation. An ideal prodrug in such applications should exhibit sufficient aqueous solubility and should be rapidly converted into the parent drug in vivo. In this regard, the most successful prodrugs of -Toc are those that exhibit sufficient water solubility and a reconversion characteristic to the parent drug after administration. We have already observed that the N,N-dimethylglycine esters of -tocopherol and vitamin K hydroquinones exhibited a significant solubility in water and have a high susceptibility to hydrolysis catalyzed by esterase in rat and human livers (15–20). In this paper, we report that the N,N-dimethylglycine ester of -Toc was synthesized and evaluated as a water-soluble prodrug of -Toc for solution formulation and, in addition, as a two-step prodrug of S--CEHC in vivo.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Melting points were determined using a Yazawa micromelting point BY-1 apparatus and are uncorrected. The microanalyses, the proton nuclear magnetic resonance spectrometry (¹H-NMR), and mass spectra measurements were carried out at the Central Microanalytical Department of Pharmaceutical Science, Fukuoka University. The ¹H-NMR spectra were recorded at 500 MHz in solutions of CDCl₃ using a JEOL JNM-A500 spectrometer. The chemical shifts are expressed in (ppm) using tetramethylsilane as the internal standard. The following abbreviations are used: s, singlet; m, multiplet. Mass spectra (MS) were obtained using a JEOL JMS-HX110 spectrometer.

A natural vitamin E mixture for food (EMix 80) and a synthesized 2,7,8-trimethyl-2-(ß-carboxylethyl)-6-hydroxychroman (racemic -CE HC) were kindly supplied by Eisai Co., Ltd. (Tokyo, Japan). Vitamin E reference standards consisting of -Toc, d-ß-tocopherol (2R,4'R,8'R-ß-tocopherol, ß-Toc), -Toc, and d--tocopherol (2R, 4'R,8'R--tocopherol, -Toc) was purchased from Eisai Co., Ltd. (Tokyo, Japan). Eserine (physostigmine hemisulfate) was purchased from Sigma Chemical Co. (St. Louis, MO). N,N-dimethylglycine hydrochloride was purchased from Tokyo Kasei Kogyo Co., Ltd. (Tokyo, Japan). HCO-60 was purchased from Nikko Chemical (Tokyo, Japan). Male Sprague-Dawley (SD) rats (210–235 g), SD rat liver microsome, and human liver microsome were purchased from Charles River, Japan, Inc. (Kanagawa, Japan). Human plasma was obtained from healthy volunteers (age 21–22) after oral informed consent. All other chemicals were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan).

Purification of d--tocopherol fraction from a natural vitamin E mixture
The EMix 80 was fractionated by flash chromatography and normal phase HPLC, and -Toc was isolated without delay by solvent evaporation under vacuum then stored under argon at -30°C. The purity of the isolated -Toc was determined by normal phase HPLC analysis comparing with -Toc, ß-Toc, -Toc, and -Toc standards, and verified by mass spectra, ¹H-NMR, and microanalysis. -Toc, and ß-Toc, and -Toc were not found in the isolated -Toc fraction. The microanalytical data agreed with the calculated values. Thus, the purity of -Toc was confirmed as the same as the -Toc standard (100%). -Toc: colorless oil; field desorption mass spectrometry (FD-MS) m/z 416 (M⁺). ¹H-NMR (CDCl₃): 6.36 (1H, s), 2.67 (2H, m), 2.13 (3H, s), 2.11 (3H, s), 1.73, (2H, m), 1.59–1.03 [24H, m, including 1.24 (3H, s)], 087–0.83 (12H, m). Anal. Calcd for C₂₈H₄₈O₂: C, 80.71; H, 11.61. Found: C, 80.77; H, 11.57.

Synthesis of d--tocopheryl N,N-dimethylglycinate hydrochloride
To a dry pyridine solution of -Toc (4.8 mmol), 5.7 mmol of N,N-dimethylglycine hydrochloride, and 5.7 mmol of dicyclohexylcarbodiimide were added. The reaction mixture was stirred at room temperature for 20 h and the dicyclohexylurea formed was removed by filtration. After the solvent was evaporated, the residue was treated with 100 ml of water and made alkaline by sodium bicarbonate. The solution was then extracted with ethyl acetate (100 ml x 3). The organic layer was dried over anhydrous sodium sulfate and evaporated. The residue was fractionated with a flash column packed Wakogel LP40, 60A using n-hexane ethyl acetate (8:2, v/v) as the eluent. The isolated ester was directly collected in isopropyl ether containing 3% HCl dioxane solution, and the precipitate was collected and recrystallized from acetone to give the hydrochloride salt of d--tocopheryl N,N-dimethylglycinate (-TDMG). It was confirmed that the synthesized -TDMG was free of -Toc (starting material) by HPLC analysis as mentioned below. White solid, Yield 83%; mp 161-163°C; FD-MS m/z: 501 (M-HCl⁺). ¹H-NMR (CDCl₃): -tocopheryl moiety 6.63 (1H, s, 5-H), 2.71 (2H, m, 4-H₂), 2.11 (3H, s, 7-CH₃), 2.02 (3H, s, 8-CH₃), 1.76, (2H, m, 3-CH₂), 1.59–1.02 [24H, m, including 1.26 (3H, s, 2-CH₃)], 0.87–0.83 (12H, m). N, N-dimethylglycine moiety 4.21 (2H, s, NCH₂CO), 3.09 [6H, s, (CH₃)₂N]. Anal. Calcd for C₃₂H₅₆NO₃Cl+0.2H₂O: C, 70.93; H, 10.49; N, 2.58. Found: C, 70.89; H, 10.54; N, 2.60.

Water solubility
The aqueous solubility of -TDMG was determined by adding 50 µmol of the ester to 1 ml of water in amber vials maintained at 25 ± 0.1°C in a constant-temperature water bath. The vials were shaken for 24 h and the contents were filtered using membrane filters (Columnguard-LCR4, 0.5 µm, Nihon Millipore Kogyo K. K., Yonezawa, Japan). The ester concentration in the filtrates was determined by the HPLC method described below. The percentage of the -TDMG in the filtrate through the membrane (0.5 µm) was above 99.7%, while the percentage of the -TDMG remaining in the filter was below 0.3%. Thus, the adherence of -TDMG in the filter was ignored.

Hydrolysis studies
The hydrolysis of the ester was studied at 37°C in an isotonic phosphate buffer (pH 7.4), rat plasma, rat liver microsome, human plasma, and human liver microsome. The stock solution of the ester was dissolved in water containing 5% methanol. The enzymatic reactions were initiated by adding 50 µl of an aqueous stock solution of the ester and 50 µl of the isotonic phosphate buffer to 900 µl of a preheated reaction medium in amber test tubes. The concentrations of the rat and human plasmas were 90%. Commercially available rat liver microsome and human liver microsome were used at 0.1 mg of protein/ml after being diluted with phosphate buffered saline (PBS). The initial concentration of the esters was 0.4–4.0 x 10^-3 M. The solutions were incubated at 37°C. At appropriate intervals, 100 µl of the reaction solution were sampled and added to 350 µl of ethanol. After 2 min of vortex mixing, followed by centrifugation at 3,000 rpm for 5 min, 50 µl of the clear supernatant was analyzed by HPLC. The initial hydrolytic rate, in units of moles of -Toc formed per liter of reaction medium volume, was calculated from the initial slope of the formation plot of -Toc versus time. No measurable chemical hydrolysis of -TDMG occurred during the time span of these hydrolysis studies in the rat plasma, the rat liver microsome, the human plasma, or the human liver microsome, as demonstrated by the HPLC results mentioned below. In the phosphate buffer, the apparent first-order rate constants for the hydrolysis were obtained by linear regression analysis of the natural logarithm of concentration versus time (correlation coefficient >0.97).

The effects of eserine on the hydrolysis of the ester in the liver microsome were also studied. The procedure for the experiment was the same as that mentioned above, except that 50 µl of eserine aqueous solution was added in place of the PBS to the liver microsome solution at the beginning of the experiment. Eserine was used at a 0–2.0 mM concentration. The studies on the hydrolysis of -TDMG and the inhibition effect by eserine have been carried out in three separate experiments.

HPLC analysis
The Shimadzu HPLC system (Kyoto, Japan) used in this study consisted of a pump (LC6A), an auto sample injector (SIL 9A), a UV detector (SPD-10AV), a spectrofluorophotometer (RF-540) equipped with a 12 µl LC flow cell, and a peak integrator (C-R7A). The eluent was spectrophotometrically monitored at 283 nm, and spectrofluorometrically at an emission of 325 nm with excitation at 298 nm. For the analysis of -TDMG and -Toc, a reversed-phase column, CAPCELL PAK C18 UG120 (4.6 x 150 mm, Shiseido, Tokyo, Japan), with a mobile phase of CH₃OH-CH₃CN (7:3, v/v) at a flow rate of 0.7 ml/min was employed. Quantitation of these compounds was achieved using linear calibration curves constructed from the peak area versus the concentration of the standard compound.

Disposition study of -TDMG and -Toc in rats
All procedures regarding animal care and use were performed in compliance with the regulations established by the Experimental Animal Care and Use Committee of Fukuoka University. The dose effect of -TDMG on the plasma disposition of -Toc was initially determined in rats. Male SD rats were fasted for 16 h prior to use, but water and sugar crystals were administered ad libitum. The solution of -TDMG was solubilized with distilled water contained 15% propylene glycol. The solution of -Toc was solubilized with water containing 10% HCO-60 and 15% propylene glycol. The drugs were administered via the left femoral vein exposed by means of a small incision under light ether anesthesia. The solution of -TDMG was injected at doses of 5, 10, and 25 mg/kg (equivalent for -Toc). The drug solution for injection was administered at 0.1 ml/100g of body weight. Blood (300 µl) was taken from the external jugular vein using heparinized syringes at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. The plasma samples (100 µl) were added to 350 µl of ethanol, vortexed for 2 min, and then centrifuged at 3,000 rpm for 5 min. The supernatant layer (50 µl) was determined by the HPLC method as mentioned above.

The rats were treated and the drugs (-TDMG and -Toc) were administered according to the procedures mentioned above. The doses of the drugs were 25 mg/kg equivalent for -Toc. Under ether anesthesia, blood (4.5 ml) was taken from the abdominal artery using a syringe containing 0.5 ml of 3.2% sodium citrate, and the liver was removed at 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. The plasma was immediately separated by centrifugation at 5°C and stored at -80°C until the HPLC analysis. The tissues were homogenized with 3 vol of 1.15% KCl solution containing 1 mM of eserine using a POLYTRON homogenizer (Kinematica, Switzerland) and stored at -80°C until the HPLC analysis. The plasma and tissue homogenated samples (100 µl) were added to 350 µl of ethanol, vortexed for 2 min and then centrifuged at 3,000 rpm for 5 min. The supernatant layer (50 µl) was determined by the HPLC method described above.

Plasma disposition of S--CEHC in rats
-TDMG or racemic -CEHC was administered to rats (8 weeks old) according to the procedures mentioned above. The solution of racemic -CEHC was solubilized with saline containing 33% polyethyleneglycol. Blood (300 µl) was taken from the external jugular vein using heparinized syringes. The plasma samples (50 µl) were subjected to the enantiometric determination of S--CEHC and R--CEHC mentioned below.

HPLC analysis of -CEHC enatiomers
The determination of -CEHC enantiomers (S--CEHC and R--CEHC) in rat plasma was carried out according to our HPLC system (14), which allowed the enantiometric determination of S--CEHC and R--CEHC without the manual isolation of pre-column fluorescent labeled -CEHC enantiomers (21). S--CEHC and R--CEHC for calibration standards were isolated from racemic -CEHC using the previously reported chiral HPLC system (21), and verified by CD spectrophotometry according to the method reported by Kantoci et al. (11). The chiral HPLC system was equipped with a chiral column Sumichiral OA-3100 (250 x 4.6 mm id, 5 mm)(Sumika Chemical Analysis Service, Co. Ltd.) and eluted with a mobile phase of 5.0 mM ammonium acetate in MeOH. The HPLC system for enantiometric determination of S--CEHC and R--CEHC consisted of three pumps, a L-7100 (Hitachi, Tokyo, Japan), two PU610-10s (Gl Science, Tokyo, Japan), two fluorescence detectors L-7480 (Hitachi), two integrators 807-IT (Jasco, Tokyo, Japan), and two 6-port valves HV-992-01 (Jasco, Tokyo, Japan). The mobile phase compositions and flow rates are as follows: H₂O-CH₃CN-TFA (650:350:1, v/v/v), 0.8 ml/min for phenyl column; H₂O-CH₃CN-TFA (400:600:1, v/v/v), 0.3 ml/min for ODS column; and CH₃OH-CH₃CN (95:5, v/v), 0.3 ml/min for chiral column. During the pretreatment procedure, briefly, -CEHC in the rat plasma (50 µl) was derivatized with a fluorescent reagent, 4-N,N-dimethylaminosulfonyl-7-piperazino-2,1,3-benzoxadiazole (DBD-PZ), and acetylated with acetyl chloride after deproteinization by adding CH₃CN-EtOH (4:1, v/v). Following purification with an Empore^TM C₁₈ cartridge, the sample was injected into a column-switching HPLC system (14) containing three different kinds of columns: TSKgel Super-Phenyl (100 x 4.6 mm, Tosoh, Tokyo, Japan), TSKgel ODS-80Ts (250 x 4.6 mm, Tosoh), and CHIRALCEL OD-RH (150 x 4.6 mm, Daicel Co. Ltd, Tokyo, Japan), which were connected through two 6-port valves equipped with a trapping column. The fraction including the -CEHC derivative, separated on the phenyl column, was introduced into the ODS column and the re-separated chiral column. The detection was made fluorometrically at 560 nm with a 450 nm excitation wavelength.

Pharmacokinetic analysis
The plasma and liver concentrations versus time data were analyzed using the model independent and statistical moment methods (22, 23). Both the maximum concentration (C_max) and its corresponding time (t_max) were obtained directly from the observed data. The systemic availability and liver availability for -Toc after iv administration of -TDMG relative to the -Toc administration was determined from the ratio of area under the concentration-time curve (AUC) of -Toc, based on Eqs. 1 and 2, respectively. The selective advantage value (24) for -Toc in the liver was calculated using Eq. 3. The systemic availability for S--CEHC after iv administration of -TDMG relative to the -Toc administration was determined from the ratio of AUC of S--CEHC based on Eq. 4.

where AUC_-Toc,-TDMG^Plasma, AUC_-Toc,-Toc^Plasma, AUC_-Toc,-TDMG^Liver, and AUC_-Toc,-Toc^Liverare the AUC values for -Toc in the plasma and tissues after the administration of -TDMG and -Toc, respectively. AUC_-TDMG,-TDMG^Plasmais the AUC value for -TDMG; D_-TDMG, D_-Toc, and D_{racemic -CEHC}are doses of -TDMG, -Toc, and racemic -CEHC, respectively; AUC_{S--CEHC,-TDMG}^Plasmaand AUC_{S--CEHC, racemic -CEHC}^Plasmaare the AUC values for the increased S--CEHC in the plasma after the administration of -TDMG and racemic -CEHC, respectively.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The -TDMG (Fig.1) was synthesized by procedures described in Materials and Methods, and characterized by mass spectrometric analysis, ¹H-NMR, and elemental analysis. The hydrochloride salt of the ester was isolated as a crystalline compound. The melting point of the ester was 161–163°C.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Chemical structures of d--Tocopherol (-Toc), 2,7,8-trimethyl-2S-(ß-carboxyethyl)-6-hydroxychroman (S--CEHC), and d--tocopheryl N,N-dimethylglycinate hydrochloride (-TDMG).

Water solubility of the -TDMG

Enzymatic hydrolysis of the -TDMG
In developing a useful prodrug, the linkage between the parent drug and the promoiety should be stable in the formulations but rapidly cleaved in vivo. The most successful prodrug of -Toc necessitates reconverting it into the parent drug by enzyme(s) encountered after administration. To determine its in vivo behavior, the kinetics of hydrolysis of the ester were investigated in an isotonic phosphate buffer (pH 7.4), rat plasma, rat liver microsome preparation, human plasma, and human liver microsome preparation at 37°C. HPLC analysis showed a significant acceleration of the hydrolytic rates of the ester to produce -Toc in rat plasma, rat liver, and human liver microsome, but not in the human plasma. The kinetics of the hydrolysis can be represented by the Michaelis-Menten model. This result obviously indicates that the hydrolysis of the ester enzymatically proceeded. The kinetic data were analyzed using the Lineweaver-Burk equation (25),

where v₀ is the initial rate of the hydrolysis, S ₀ is the initial concentration of the ester, K_m is the Michaelis constant, and V_max is the maximal hydrolytic rate for a saturating substrate concentration at a given enzyme concentration.

Representative Lineweaver-Burk plots are shown in Fig. 2 , which depict the hydrolysis in the rat and human liver microsome preparations. The kinetic parameters of V_max and K_m generated from the initial-rate data and a linear regression analysis of Eq. 5 are listed in Table 1 along with V_max/K_m.

View larger version (15K): [in this window] [in a new window]   Fig. 2. Representative Lineweaver-Burk plots of initial rates for the hydrolysis of -TDMG in the rat liver microsome and the human liver microsome at pH 7.4 and 37°C. Open circle, rat liver microsome; closed circle, human liver microsome. Each point represents the mean ± SD of three experiments. The lines represent least-square regression lines (r > 0.98).

View this table: [in this window] [in a new window]   TABLE 1. Kinetic parameters for the hydrolysis of -TDMG in vitro at pH 7.4 and 37°C

Considering the therapeutic application of the ester as a prodrug, it is an important criteria that the ester can be readily cleaved by a human enzyme. A significant acceleration of the hydrolytic rate of the ester was found in the human liver microsome preparation, but not in the human plasma preparation (Table 1). This in vitro result suggested that the hydrolysis of the ester could be catalyzed by enzyme(s) located in humans. In humans, the hydrolysis of -TDMG has been studied only in vitro.

As described above, -TDMG was mainly cleaved by an enzyme contained in the liver to release the parent drug. To assess whether the observed catalytic cleavage of -TDMG in the liver could be attributed to liver esterase, we investigated the effects of eserine, an esterase inhibitor, on the release of -Toc in the rat and human liver microsome preparations. The catalytic hydrolysis of -TDMG by both rat and human liver microsomes was prominently inhibited in the presence of eserine (Fig. 3) . It has been postulated that carboxylesterase is present in both the rat (26) and human (27) liver microsomes and that eserine is an inhibitor of carboxylesterase (28). Thus, the results suggested that the rat and human liver carboxylesterases mainly catalyzed the reconversion of the prodrug.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Effect of eserine on the rat liver and the human liver enzymatic hydrolysis of -TDMG. Open circle, in the rat liver microsome; closed circle, in the human liver microsome. The initial concentration of -TDMG was adjusted at 0.4 mM. Each point represents the mean ± SD of three experiments.

For the purpose of developing the prodrug for a solution formulation, a prodrug with a high water solubility and a high reconversion rate into -Toc appeared the most promising for further in vivo studies. -TDMG would be a suitable water-soluble prodrug of -Toc for iv administration, because -TDMG was a crystalline compound and soluble in water (over 50 mM) and -TDMG is converted into -Toc catalyzed by esterases in both the rat and human livers. Based on these findings, further in vivo studies are now being carried out.

Disposition of the prodrug after iv administration in rats
The dose effect of -TDMG on the plasma disposition of -Toc was preliminarily determined in rats in the dose range from 5 to 25 mg/kg equivalents to -Toc (Fig. 4) . The rapid appearance and dose dependently increased level of -Toc in plasma after -TDMG administration indicated that -Toc was regenerated from -TDMG in vivo. The AUC values of -Toc from 0 to 24 h after the administration of -TDMG were also increased dose dependently and a linear correlation was found between the AUC of -Toc and the dose of -TDMG (data not shown). This result indicated that there was no saturated process during the regeneration of -Toc in the dose range tested in this study. Thus, further studies were carried out using a dose of 25 mg/kg equivalent to -Toc.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Mean plasma concentration of -Toc after the intravenous administration of -TDMG in the rats. The doses are 25 mg/kg (open circle), 10 mg/kg (closed circle), 5 mg/kg (square) equivalent for -Toc. Each point represents the mean ± SD of three rats.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Mean plasma and liver concentration of -TDMG and -Toc after the intravenous administration of -TDMG and -Toc in the rats. Open circle, -TDMG; closed circle, -Toc after -TDMG administration; square -Toc after -Toc administration. Each point represents the mean ± SD of three rats. The dose is 25 mg/kg equivalent for -Toc.

View this table: [in this window] [in a new window]   TABLE 2. Pharmacokinetic parameters in plasma and liver after the intravenous administration of -Toc and -TDMG in the ratsa

These disposition studies clearly indicated that -TDMG would be a useful candidate for the parenteral prodrug of -Toc. The effective delivery of -Toc with the iv administration of -TDMG is a meaningful method for achieving a rapid and accurate onset of action of -Toc and might alter the several prospective biological activities of -Toc. In preliminary experiments, it was found that the iv administration of the prodrug to the middle cerebral artery occlusion of mice afforded a lesser degree of cerebral brain damage compared to administration of -Toc solubilized in DMSO. The results of the preventative effect in cerebral infarction will be the subject of a subsequent paper.

Plasma disposition of S--CEHC after iv administration of the prodrug
Since S--CEHC contains the same cromanol structure as -Toc, S--CEHC is readily oxidized by atmospheric oxygen as well as -Toc. S--CEHC exhibits a 20-fold more potent natriuretic activity than R--CEHC (12), but shows a rapid elimination rate after iv administration (14). These characteristics limit the therapeutic applications of S--CEHC. It has been shown that -Toc is mainly metabolized to S--CEHC without epimerization at C2 (10, 11), and the conversion to S--CEHC was catalyzed by cytochrome P450 3A (CYP3A) in a cell culture (31). Therefore, it seems that the use of -TDMG as a two-step prodrug for S--CEHC is a meaningful method for overcoming the delivery problems of S--CEHC because -TDMG can efficiently deliver -Toc to the liver.

To evaluate the -TDMG as a two-step prodrug of S--CEHC, the plasma disposition of S--CEHC after the iv administration of -TDMG was compared with that of the racemic -CEHC. As a result of the HPLC analysis, only S--CEHC was detected in the plasma during the experiment interval after the -TDMG administration (Fig. 6) . The time-course of the plasma concentration of S--CEHC is shown in Fig. 7 . The pharmacokinetic parameters for S--CEHC are summarized in Table 3. Following the iv administration of -TDMG, the plasma S--CEHC level was rapidly increased and achieved the maximum accumulation at 1 h after the administration, and the mean residence time (MRT) of S--CEHC was prolonged by eight times compared to the racemic -CEHC administration. The relative systemic availabilities for S--CEHC (F) after the -TDMG and racemic -CEHC administration have equivalent values of 102% and 100%, respectively. This result clearly indicated that -TDMG can act as a two-step prodrug of S--CEHC. Although the mechanism for the delivery of S--CEHC with -TDMG administration could not be confirmed, the selective appearance of S--CEHC suggested that -TDMG was first reconverted to -Toc and metabolized to S--CEHC. It has already been shown that -Toc was metabolized to -CEHC by CYP3A (31). Thus, when -TDMG is taken together with drugs that are principally metabolized by CYP3A, the disposition kinetics of -CEHC after -TDMG administration may be altered due to competitive inhibition between -Toc and the drugs on the CYP3A.

View larger version (17K): [in this window] [in a new window]   Fig. 6. Representative chromatograms of enantiometric separation of fluorescent -CEHC derivative. A: Racemic -CEHC standard. B: Rat plasma sample at 1 h after iv administration of -TDMG.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Mean plasma concentration of S--CEHC after the intravenous administration of -TDMG and racemic -CEHC in the rats. Circle, -TDMG; square, racemic -CEHC. Each point represents the mean ± SD of four rats. The doses are shown in Table 3.

View this table: [in this window] [in a new window]   TABLE 3. Pharmacokinetic parameters for S--CEHC after iv administration of -TDMG and racemic -CEHC in the rats

	ACKNOWLEDGMENTS

 
A part of this work was supported from the Grant-in-Aid for Encouragement of Young Scientists (KAKENHI 14771346) from The Ministry of Education, Culture, Sports, Science and Technology (MEXT) granted to K.M.

Submitted on July 26, 2002
Revised on September 6, 2002

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

1. Lehmann, J., H. L. Martin, E. L. Lashley, M. W. Marshall, and J. T. Judd. 1986. Vitamin E in foods from high and low linoleic acid diets. J. Am. Diet. Assoc. 86: 1208–1216.[Medline]

2. Bieri, J. G., and R. P. Evarts. 1974. Vitamin E activity of -tocopherol in the rat, chick and hamster. J. Nutr. 104: 850–857.

3. Ohrvall, M., G. Sundlof, and B. Vessby. 1996. Gamma, but not alpha, tocopherol levels in serum are reduced in coronary heart disease patients. J. Intern. Med. 239: 111–117.[CrossRef][Medline]

4. Kontush, A., T. Spranger, A. Reich, K. Baum, and U. Beisiegel. 1999. Lipophilic antioxidants in blood plasma as markers of atherosclerosis: the role of -carotene and -tocopherol. Atherosclerosis. 144: 117–122.[CrossRef][Medline]

5. Kristenson, M., B. Zieden, Z. Kucinskiene, L. S. Elinder, B. Bergdahl, B. Elwing, A. Abaravicius, L. Razinkoviene, H. Calkauskas, and A. G. Olsson. 1997. Antioxidant state and mortality from coronary heart disease in Lithuanian and Swedish men: concomitant cross sectional study of men aged 50. Br. Med. J. 314: 629–633.[Abstract/Free Full Text]

6. Cooney, R. V., A. A. Franke, P. J. Harwood, V. Hatch-Pigott, L. J. Custer, and L. J. Mordan. 1993. -Tocopherol detoxification of nitrogen dioxide: superiority to -tocopherol. Proc. Natl. Acad. Sci. USA. 90: 1771–1775.[Abstract/Free Full Text]

7. Cooney, R. V., P. J. Harwood, A. A. Franke, K. Narala, A-K. Sundström, P-O. Berggren, and L. J. Mordan. 1995. Products of -tocopherol reaction with NO₂ and their formation in rat insulinoma (RINm5F) cells. Free Radic. Biol. Med. 19: 259–269.[CrossRef][Medline]

8. Christen, S., A. A. Woodall, M. K. Shigenaga, P. T. Southwell-Keely, M. W. Duncan, and B. N. Ames. 1997. -Tocopherol traps mutagenic electrophiles such as NOx and complements -tocopherol: physiological implications. Proc. Natl. Acad. Sci. USA. 94: 3217–3222.[Abstract/Free Full Text]

9. Hoglen, N. C., S. C. Waller, I. G. Sipes, and D. C. Liebler. 1997. Reactions of peroxynitrite with gamma-tocopherol. Chem. Res. Toxicol. 10: 401–407.[CrossRef][Medline]

10. Wechter, W. J., D. Kantoci, E. D. Murray, Jr., D. C. D'Amico, M. E. Jung, and W-H. Wang. 1996. A new endogenous natriuretic factor: LLU-. Proc. Natl. Acad. Sci. USA. 93: 6002–6007.[Abstract/Free Full Text]

11. Kantoci, D., W. J. Wechter, E. D. Murray, Jr., S. A. Dewind, D. Borchardt, and S. I. Khan. 1997. Endogenous natriuretic factor 6: the stereochemistry of a natriuretic -tocopherol metabolite LLU-. J. Pharmacol. Exp. Ther. 282: 648–656.[Abstract/Free Full Text]

12. Murray, E. D., Jr., W. J. Wechter, D. Kantoci, W-H. Wang, T. Pham, D. D. Quiggle, K. M. Gibson, D. Leipold, and B. M. Anner. 1997. Endogenous natriuretic factors 7: biospecificity of a natriuretic -tocopherol metabolite LLU-. J. Pharmacol. Exp. Ther. 282: 657–662.[Abstract/Free Full Text]

13. Jiang, Q., I. Elson-Schwab, C. Courtemanche, and B. N. Ames. 2000. -Tocopherol and its major metabolite, in contrast to -tocopherol, inhibit cyclooxygenase activity in macrophages and epithelial cells. Proc. Natl. Acad. Sci. USA. 97: 11494–11499.[Abstract/Free Full Text]

14. Hattori, A., T. Fukushima, K. Hamamura, M. Kato, and K. Imai. 2001. A fluorimetric, column-switching HPLC and its application to an elimination study of LLU- enantiomers in rat plasma. Biomed. Chromatogr. 15: 95–99.[CrossRef][Medline]

15. Takata J., Y. Karube, Y. Nagata, and Y. Matsushima. 1995. Prodrug of vitamin E. 1. preparation and enzymatic hydrolysis of aminoalkanecarboxylic acid esters of d--tocopherol. J. Pharm. Sci. 84: 96–100.[CrossRef][Medline]

16. Takata, J., S. Ito, Y. Karube, Y. Nagata, and Y. Matsushima. 1997. Water-soluble prodrug of vitamin E for parenteral use and its effect on endotoxin-induced liver toxicity. Biol. Pharm. Bull. 20: 204–209.[Medline]

17. Takata, J., Y. Karube, M. Hanada, K. Matsunaga, Y. Matsushima, T. Sendo, and T. Aoyama. 1995. Vitamin K prodrugs: 1. synthesis of amino acid esters of menahydroquinone-4 and enzymatic reconversion to an active form. Pharm. Res. 12: 18–23.[CrossRef][Medline]

18. Takata, J., Y. Karube, M. Hanada, K. Matsunaga, Y. Matsushima, T. Sendo, and R. Oishi. 1995. Vitamin Kprodrugs: 2. water-soluble prodrugs of menahydroquinone-4 for systemic site-specific delivery. Pharm. Res. 12: 1973–1979.[CrossRef][Medline]

19. Takata, J., Y. Karube, M. Hanada, K. Matsunaga, and H. Iwasaki. 1999. Prodrug for bioreductive activation-independent delivery of menahydroquinone-4: Human liver enzymatic activation and its action in warfarin-poisoned human liver. Biol. Pharm. Bull. 22: 172–178.[Medline]

20. Takata, J., Y. Karube, K. Matsunaga, M. Hanada, R. Hidaka, and Y. Matsushima. 1999. Prodrugs for systemic bioreductive activation-independent delivery of phyllohydroquinone, an active form of phylloquinone (vitamin K1) 1: preparation and in vitro evaluation. Biol. Pharm. Bull. 22: 1347–1354.[Medline]

21. Hattori A., T. Fukushima, and K. Imai. 2000. Occurrence and determination of a natriuretic hormone, 2,7,8-trimethyl-2-(ß-carboxyethyl)-6-hydroxy chroman, in rat plasma, urine, and bile. Anal. Biochem. 281: 209–215.[CrossRef][Medline]

22. Yamaoka, K., Y. Tanigawara, T. Nakagawa, and T. Uno. 1981. A pharmacokinetic analysis program (MULTI) for microcomputer. J. Pharmacobiodyn. 4: 879–885.[Medline]

23. Yamaoka K., T. Nakagawa, and T. Uno. 1978. Statistical moments in pharmacokinetics. J. Pharmacokinet. Biopharm. 6: 547–558.[CrossRef][Medline]

24. Eriksson, U. G., and T. N. Tozer. 1987. Pharmacokinetic evaluation of regional drug delivery. Acta Pharm. Jugosl. 37: 331–344.

25. Lineweaver, H., and D. Burk. 1934. The determination enzyme dissociation constants. J. Am. Chem. Soc. 56: 658–666.[CrossRef]

26. Hosokawa, M., T. Maki, and T. Satoh. 1987. Multiplicity and regulation of hepatic microsomal carboxylesterases in rats. Mol. Pharmacol. 31: 579–584.[Abstract]

27. Hosokawa, M., T. Maki, and T. Satoh. 1990. Characterization of molecular species of liver microsomal carboxylesterases of several animal species and humans. Arch. Biochem. Biophys. 277: 219–227.[CrossRef][Medline]

28. La Du, B. N., and H. Snady. 1971. Esterases of human tissue. In Handbook of Experimental Pharmacology Concepts in Biochemical Pharmacology. Part 2. B. B. Brodie and J. R. Gillette, editors. Springer-Verlag, Berlin, Hidelberg, New York. 447–479.

29. Bundgaard, H., C. Larsen, and P. Thorbek. 1984. Prodrug as drug delivery system XXVI. Preparation and enzymatic hydrolysis of various water-soluble amino acid esters of metronidazole. Int. J. Pharm. 18: 67–77.

30. Bundgaard, H., and E. Falch. 1985. Allopurinolprodrugs. III. Water-soluble N-acyoxymethyl allopurinol derivatives for rectal or parenteral use. Int. J. Pharm. 25: 27–39.

31. Parker, R. S., T. J. Sontag, and J. E. Swanson. 2000. Cytochrome P4503A-dependent metabolism of tocopherols and inhibition by sesamin. Biochem. Biophys. Res. Commun. 277: 531–534.[CrossRef][Medline]

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